N-methyl-D-aspartate (NMDA) glutamate receptor-ion channels in the brain are important mediators of the behavioral effects of alcohol. Although alcohol is known to inhibit NMDA receptors by influencing ion channel gating, its molecular site of action and the mechanism underlying this effect have not been established. Previous studies have pointed to a role of the membrane-spanning domains of neurotransmitter-gated ion channels in the actions of alcohol. Results from this laboratory have shown that a conserved methionine residue (Met823) in the fourth membrane-associated (M) domain of the NMDA receptor NR2A subunit profoundly influences ion channel gating. Recent evidence obtained in this laboratory has shown that NR2A(Met823) also affects alcohol sensitivity of the receptor in a manner that is consistent with a site of alcohol action, and that adjacent residues may also influence both ion channel gating and alcohol sensitivity. The main hypothesis to be tested in the proposed studies is that this residue and adjacent residues that are also involved in ion channel gating form a key site of alcohol action on the NMDA receptor-ion channel. These studies will incorporate whole-cell and single-channel patch-clamp electrophysiological recording to investigate the following specific aims: 1) the role of domains adjacent to NR2A(Met823) in the regulation of NMDA receptor ion channel gating and alcohol sensitivity;2) whether these regions constitute a key site of alcohol action on the NMDA receptor;3) the precise kinetic mechanism by which alcohol interacts with these sites to alter NMDA receptor ion channel gating;and 4) whether effects of NMDA receptor mutations on gating kinetics and alcohol modulation observed in a non-neuronal cell line are also observed in CNS neurons. The results of these studies will yield important information about the molecular mechanism of alcohol modulation of the NMDA receptor, new insights into the structure and function of the NMDA receptor-ion channel, and a better understanding of the ways in which alcohols and similar molecules interact with proteins in general. Because NMDA receptors have vital and widespread roles in cognition, motor function, and memory, and contribute importantly to both the subjective effects of alcohol as well as its CNS depressant effects, the information gained in these studies will represent an important step forward in the understanding of alcohol effects on the nervous system.